Quantitation of peptides and proteins by matrix‐assisted laser desorption/ionization mass spectrometry using 18O‐labeled internal standards

Mirgorodskaya, O. A.; Kozmin, Yuri; Титов, М. И.; Körner, Roman; Sönksen, Carsten P.; Roepstorff, Peter

doi:10.1002/1097-0231(20000730)14:14<1226::aid-rcm14>3.3.co;2-m

Cited by 163 publications

(239 citation statements)

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“…To this end, differential techniques that use stable isotope incorporation into peptides should have been employed. 40,41 Nevertheless, MALDI-MS analysis of these protein bands did not reveal qualitative differences for these more intensely stained proteins in the case of supernatant from untreated mitochondria. Hence it is possible that a tBid-dependent release of proteases (such as Omi) partially degrades proteins in the mitochondrial supernatant, resulting in a lesser protein content.…”

Section: Resultsmentioning

confidence: 99%

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

et al. 2002

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A crucial event in the process of apoptosis is caspasedependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization postsource decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondriaderivedfrom liver-specificBcl-2-transgenicmice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/ Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

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Section: Resultsmentioning

confidence: 99%

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid

et al. 2002

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“…Some techniques introduce the label in viable cells in cell culture ( 14 N/ 15 N labeling [122] and stable isotope labeling by amino acids in cell culture, SILAC [123,124]). Others introduce the tag at the protein level (isotope-coded affinity tags, ICAT [125]) or peptide level (isobaric peptide tags for relative and absolute quantifi-cation, iTRAQ [126] or 18 O-labeling during tryptic digestion [127]), whereas yet others use a label-free strategy in which quantitative information is extracted from LC-MS (extracted ion current, XIC) or MS/MS data (protein abundance index, PAI) in sequential experiments [128][129][130]. Not all quantitation strategies are efficient for phosphoproteomic studies.…”

Section: Quantitation Strategies For Phosphoproteomic Studiesmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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“…Desiderio and Kai employed enzyme-catalyzed 18 O exchange for the preparation of internal standards for MS-based quantitation of peptides in biological extracts [8]. Mirgorodskaya et al and Stewart et al [9,10] proposed the use of 16 O/ 18 O labeling for MS-based quantitation of proteins; the application of this technique as an effective quantitative solutionbased, shotgun proteomic tool was first reported by Yao et al [5]. Coupling the SDS-PAGEbased quantitative approach with post-digestion 18 O exchange for differential proteomics of protein complexes was first proposed by Bantscheff etal.…”

Section: Introductionmentioning

confidence: 99%